The extraction and analysis of flavonoid compounds were carried out as described by Li et al. [29 (link)]. Briefly, the frozen tissue powder (0.5 g) was ground in 1.5 mL phenolic compound, extracting a solution containing 70% methanol and 2% formic acid at 0–4 °C. After centrifugation at 10,000 g for 20 min, the supernatant was passed through a 0.22-μm syringe filter prior to analysis.
The Inertsil ODS-3 column (5.0 μm particle size, 4.6 mm × 250 mm, GL Sciences Inc., Tokyo, Japan) was used for the separation, preceded by an Inertsil ODS-3 Guard Column. A 10-μL sample of the filtered supernatant was injected into an LC-20A Liquid Chromatograph with a diode array detector (Shimadzu Corporation, Tokyo, Japan). Solvent A consisted of 10% formic acid (11.36% 88% formic acid) in water and solvent B consisted of 10% formic acid and 1.36% water (11.36% 88% formic acid) in acetonitrile. The gradients used were 95% A (0 min), 85% A (25 min), 78% A (42 min), 64% A (60 min), and 95% A (65 min) with a post-run time of 10 min. The flow rate was 1.0 mL min−1, and the column temperature was set at 35 °C. Flavonoid compounds were detected at 365 nm for flavonols and 520 nm for anthocyanins.
The Inertsil ODS-3 column (5.0 μm particle size, 4.6 mm × 250 mm, GL Sciences Inc., Tokyo, Japan) was used for the separation, preceded by an Inertsil ODS-3 Guard Column. A 10-μL sample of the filtered supernatant was injected into an LC-20A Liquid Chromatograph with a diode array detector (Shimadzu Corporation, Tokyo, Japan). Solvent A consisted of 10% formic acid (11.36% 88% formic acid) in water and solvent B consisted of 10% formic acid and 1.36% water (11.36% 88% formic acid) in acetonitrile. The gradients used were 95% A (0 min), 85% A (25 min), 78% A (42 min), 64% A (60 min), and 95% A (65 min) with a post-run time of 10 min. The flow rate was 1.0 mL min−1, and the column temperature was set at 35 °C. Flavonoid compounds were detected at 365 nm for flavonols and 520 nm for anthocyanins.